The functions of endothelial cell derived nitric oxide (NO) continue to expand following the Nobel Prize-winning discovery of nitric oxide as a signaling molecule in the cardiovascular system. While the molecular mechanisms for the regulation of vascular tone by NO are well appreciated, the mechanisms by which nitric oxide regulate metabolic processes remain unclear. During the last funding period we developed and implemented novel mass spectrometry-based proteomic technologies that enable the precise mapping of protein S-nitrosocysteine residues in vivo. The data collected from wild type and endothelial nitric oxide synthase null (eNOS-/-) mouse organs revealed selective and reversible S-nitrosylation of key regulatory proteins in metabolic pathways implying a novel biochemical and molecular mechanism by which eNOS-NO regulate metabolic processes. The current proposal extends these observations by proposing to study the mechanisms by which eNOS-NO and selective protein S-nitrosylation affords metabolic flexibility. Metabolic flexibility is a response that enables organisms to adapt to metabolic needs such as nutrient deprivation or nutrient excess. The molecular mechanisms of adaptation to nutrient excess are becoming increasingly important since metabolic syndrome, obesity and resulting cardiovascular complications are major health issues in the United States. Based on the premise that NO availability declines in metabolic syndrome, clinical trials are recruiting subjects aiming to corrct the NO deficiency and thus improve cardiovascular and metabolic phenotypes. To provide mechanistic insights and potentially identify salutary downstream metabolic responses, we will test novel therapeutic strategies that increase the bioavailability of NO in genotypically and phenotypically well-characterized mouse models that replicate many features of the metabolic syndrome. The goals of this application are twofold: (1) Determine the contribution of eNOS/nitric oxide and selective protein S-nitrosylation signaling in metabolic adaption during dynamic changes in nutrient status. (2) Evaluate if novel strategies that increase the bioavailability of NO will restore NO and selective protein S-nitrosylation signaling to reverse th metabolic phenotypes of eNOS-NO deficient mice. Completion of the proposed work will: (i) provide new mechanistic insights regarding the contribution of eNOS-NO and selective protein S-nitrosylation in the coordination of metabolic activity and flexibility. (ii) Provide a foundatio of knowledge that is translational and directly transferable to ongoing clinical studies. There is a clear need for the development of therapeutics as well as necessity for gaining mechanistic understanding of the processes that cause metabolic failure. This area of research is now at exciting crossroads, because it represents the confluence of clinical trials, translational researc in animal models that replicate features of the human disease and the fields of NO biological chemistry, lipid biology, proteomics and metabolism.